Cystic fibrosis (CF) is a disease that affects over 11,000 people in the UK. It is a genetic condition that affects many organs including the lungs, pancreas, kidneys and liver. New drugs called "modulators" have meant people with CF are now living much longer. Until recently, heart disease was rare in CF, but with the new modulators there are increasing concerns that heart disease may become a big problem in the future. This is partly to do with the drugs causing weight gain and higher blood pressure, which are risk factors for heart disease.

My PhD project aims to find out whether the blood vessels and hearts of people with CF are healthy or diseased. I will then find out how the blood vessels are changing over time and work out what things are driving those changes.

I will measure the health of the blood vessels and heart using an ultrasound machine to understand what the pattern of disease is like and who might be at the highest risk for heart disease in the future. I will then repeat these measurements a year later. I will compare people of different ages and with different types of disease to understand what things may help us identify heart disease as soon as possible.

In the general population, doctors often use medical prediction tools to find out who is at the highest risk for heart disease. We do not know if these work for people with CF, so I will also find out whether those prediction tools are useful in CF.

It is vital to understand who may be at risk for heart disease, as one of the most effective ways of treating heart disease is to prevent it from happening. This work may pave the way for future studies to test early treatment for heart disease in those people we identify might be at high risk. Early prevention treatment could reduce the risk of heart disease and ultimately improve the length and quality of life of people living with CF. This is particularly important given people with CF already have a much shorter life expectancy than the general population.

In summary, this PhD project will help improve our understanding of heart disease in CF and help identify the best way forward to prevent heart disease causing health problems related to heart disease for these individuals in the future.

Background:

Cystic fibrosis (CF) is an autosomal recessive disorder characterised by a chloride ion transport defect, leading to dehydrated epithelial secretions, resulting in an inflammatory/infection cycle. This manifests itself most obviously in the lungs but affects multiple organ sites.

Before the development of Cystic Fibrosis Transmembrane Receptor (CFTR) modulators, the mainstay of disease was supportive with a focus on airway clearance, aggressive treatment and prevention of infection and optimisation of nutrition and other common co-morbidities like cystic fibrosis diabetes (CFD) . As a result of the introduction of highly effective modulator therapies (HEMT), overall wellbeing and prognostic outlook has improved significantly. These recent step-changes in prognostic outlook come after small but persistent improvements over the last three decades, such that prior to the introduction of CFTR modulator therapy, the median life expectancy had reached approximately 50 years of age, but now expected it to be longer. The latest HEMT in the market is Elexacaftor/Tezacaftor/Ivacaftor (ETI). Licensed in 2020, it is a ground-breaking development of treatment. About 90% of people with CF worldwide are eligible for the medication according to their genotypes, drastically improving these people's intra- and extra-pulmonary outcomes.

Cardiovascular disease (CVD) is the leading cause of death globally [5]. CVD prevention is essential as there are modifiable risk factors that can be controlled and reducing the occurrence of cardiovascular disease morbidity and mortality. Although people with CF possess numerous traditional CVD risk factors, including a high-fat-high-calorie diet and CF diabetes, historically very few cases are presented with cardiovascular disease in CF. PwCF were considered not at high risk of CVD development because of the shorter life expectancy, low body mass index (BMI) and low blood pressure. However, the risk profile is changing rapidly for people receiving ETI and there are concerns there is likely to be a "wave" of CVD in people living with CF in the coming decade. Some of the rationale for these concerns are set out below.

PwCF were generally considered at risk of malnutrition and being underweight and were therefore recommended to have a high-energy, low-nutrient dietary intake. The introduction of ETI improves nutritional absorption, and as a consequence the weight and BMI are also improved. Unlike lung function improvements that often peaked within eight weeks of ETI initiation, clinical trial data and real-world studies show that increased weight and BMI do not stop increasing. In clinical trials of HEMT, rapid weight gain was commonly observed, and the mean BMI increased by 1.04 kg/m2 in just 24 weeks. The ongoing study showed progressive weight gain continuing two years after HEMT initiation, with the mean BMI increased by 1.6kg/m2. In adults, the mean BMI is around 25, suggesting that approximately half of pwCF receiving HEMT are overweight. Data shows that despite reducing dietary energy intake, BMI has not proportionally decreased, which suggests that metabolic changes also have a significant role in weight and BMI in pwCF on ETI.

It is known that pwCF have lower blood pressure compared to the general population, with a tendency for blood pressure to increase less with age. This is thought to be caused by greatly increased sweat chloride loss. With HEMT initiation, the chloride channels are theoretically fixed and therefore, blood pressure could be increased. There is evidence that the prevalence of hypertension in pwCF has increased, with case series reported in Italy and a US study with a 30% increase in hypertension prevalence in a single CF centre.

Arterial stiffness is also recognised as a highly clinically relevant and independent prognostic biomarker. Arterial stiffness explains the inter-relations between the blood flow and the arterial wall tissue. Arteries become stiffer with ageing and particular diseases, and as in other chronic diseases, increased arterial stiffness and endothelial dysfunction have been reported in CF in a few studies. All of these were, however, conducted prior to the widespread availability of modulator therapy. It is therefore essential to understand the progress of vascular health in people with CF in the HEMT era and identify future research targets. If this is a growing trend, this increase in blood pressure and arterial stiffness will likely increase the risk for CVD morbidity and mortality.

Atherosclerosis is a process underlying many cardiovascular diseases; It is the build-up of fats, cholesterol and other substance at the artery walls. The growth of plaques can occlude or rupture the arteries, causing a cardiovascular event. Atherosclerosis and arterial stiffness often co-exist and exacerbate each other's effects. Atherosclerotic plaques contribute to arterial stiffness by increasing the stiffness of the vessel wall and reducing its ability to expand in response to changes in blood pressure. Conversely, arterial stiffness promotes the development and progression of atherosclerosis by increasing shear stress on the endothelium, a single-cell layer that lines the cardiovascular system, which is responsible for maintaining the tone and health of blood vessels. This promotes endothelial dysfunction.

Endothelial dysfunction can be initiated by exposure to numerous risk factors including hypertension and inflammation, causing damages to the arterial cell wall, and is considered an early marker of atherosclerosis. Dysfunctional endothelial cells exhibit impaired nitric oxide (NO) production, increased expression of adhesion molecules, and enhanced oxidative stress, promoting the adhesion of circulating leukocytes and the infiltration of the low-density lipoprotein cholesterol (LDL-C) into the arterial wall. These processes initiate the inflammatory cascade that drives the formation of atherosclerotic plaques. There is evidence of vascular endothelial dysfunction in children and young adults with CF compared to the normal cohort.

Chronic inflammation is another risk factor of CF which may predispose people to CVD. Other chronic inflammatory diseases such as systemic lupus erythematosus (SLE), HIV infection and rheumatoid arthritis (RA) are associated with increased cardiovascular morbidity and mortality. A recent multinational study suggested that pwCF are already at equivalent CVD risk to those living with HIV, SLE or RA. Some of the pathophysiology relevant to these diseases is shared by CF, for example, NETosis and tumour necrosis factor. They are common inflammatory features of CF and are implicated in atherogenesis and heart failure. More specifically, chronic inflammation diseases are associated with subclinical atherosclerosis prevalence as it contributes to endothelial dysfunction. Inflammation is also key in all stages of atherosclerotic process including vascular lesion formation, which is also exacerbated by other risk factors.

Impaired glycaemic control is another significant risk factor precipitating cardiovascular events; over one-third of people with CF aged 16 and over are being treated with CF diabetes. HEMT has been associated with improved glycaemic control and reduced glycaemic variability; Diabetes, nonetheless, does not appear to be routinely improved. A US study shows an equal proportion of noted deterioration or improvement in glycaemic control after HEMT initiation. Therefore, this well-known risk factor for CVD may continue to be apparent after HEMT.

Given that CVD risk factors are becoming more prevalent, it is not unreasonable to assume people with Cystic Fibrosis are at risk of developing cardiovascular disease. I have evaluated the QRISK3 score, a general population CVD risk prediction tool, in the Merseyside and North Wales adult CF population with CF diabetes and found that a 1-year post-HEMT initiation was associated with over 20% relative increase in 10-year CV risk. This was the first study of QRISK in the post-modulator era. QRISK has previously performed sub-optimally in other inflammatory conditions but to date, no studies have evaluated its performance in CF. To address the above uncertainties on vascular risk profiles in pwCF, a more in depth understanding of vascular health is needed to determine the actual cardiovascular health in pwCF. A priority is to evaluate the atherosclerosis development in pwCF, which can be demonstrated by endothelial dysfunction and arterial stiffness. Common inflammatory disease listed above have their own modifying score on QRISK3 and we predict CF should have their own modifying score as those common inflammatory disease.

To date there is little to no identification of overall cardiovascular disease risk in pwCF following the advent of HEMT. Despite the transformative impact of HEMT on pulmonary function and life expectancy in CF patients, there remains a critical gap in understanding the potential cardiovascular implications of these therapies. Given the emergence of new risk factors associated with HEMT, such as significant weight gain and alterations in blood pressure dynamics, there is an urgent need for early and thorough assessment of markers indicative of cardiovascular health in this population. This could include blood pressure, arterial stiffness, endothelial dysfunction, weight/BMI, inflammatory profiles, and diabetes/blood lipid profiles. By integrating these multidimensional assessments into routine clinical care for individuals with CF, healthcare providers can proactively identify and manage cardiovascular risk factors, thus promoting optimal cardiovascular health outcomes in this population.

STUDY:

This study involves three parts (WP1, WP2, WP3). Work Package 1: Cross-sectional analysis of CV health in PwCF and age- and sex-matched control Work Package 2: Longitudinal analysis of CV health in PwCF and age-matched control Work Package 3: Evaluation of the relationship between CVD risk prediction tools and CV health in pwCF

WORK PACKAGE 1 AND 2

The following will be assessed:

• Arterial stiffness (PWV)

• Endothelial-mediated function (FMD)

• Blood-borne biomarkers

  • Full blood count (FBC)

  • Renal function

  • Glycated haemoglobin (HbA1c)

  • Full lipid profile

    • Total Cholesterol
    • High-density lipoprotein cholesterol (HDL-C)
    • Low--density lipoprotein cholesterol (LDL-C)
    • Triglycerides

  • Inflammatory markers

    • C-Reactive Protein (CRP)
    • Creatine Kinase-MB (CKMB)

  • Resting blood pressure, measured at supine and upright position

• Sweat Chloride levels

• Height and weight (for BMI measurement)

• Body Composition QRISK3 related data will be collected as well (https://qrisk.org/three/index.php). This includes the following additional clinical information - ethnicity, angina or heart attack in a 1st degree relative <60; history of migraines, rheumatoid arthritis, systemic lupus erythematosus or severe mental illness; taking atypical antipsychotic medication, diagnosis of, or treatment for erectile dysfunction.

Participants will be reminded not to use any short-acting bronchodilators for at least 6 hours, not to have any high fat diet for at least 12 hours, no alcohol and exercise for at least 24 hours and no vitamin supplements for at least 72 hours before attending for assessment. Following baseline measurements, participants will have all measurements repeated at 12 months. Participants will have routine care as managed by their general practitioner and the Cystic Fibrosis team.

MEASUREMENT OF ARTERIAL STIFFNESS Arterial stiffness will be assessed using Pulse Wave Velocity (PWV) and Augmentation Index (AIx)-two widely validated non-invasive markers of large artery stiffness and wave reflection, respectively.

Pulse wave velocity (PWV) will be estimated using an IEM Mobil-O-Graph, which analyses oscillometric pulse waveforms recorded at the brachial artery. Using the ARCSolver algorithm, it provides an estimate of central (aortic) PWV. Participants will rest in a supine position for at least 15 minutes in a quiet, temperature-controlled room prior to measurement. Measurements will be taken in duplicate, and the average value will be used for analysis.

AIx will be estimated from the brachial pulse waveform recorded via the oscillometric Mobil-O-Graph device. Central aortic waveforms are reconstructed using the ARCSolver algorithm, and AIx is calculated as the difference between the second and first systolic peaks, expressed as a percentage of pulse pressure. To minimise the influence of inter-individual heart rate variability, values will be normalised to a heart rate of 75 bpm (AIx@75).

MEASUREMENT OF ENDOTHELIAL FUNCTION Endothelial function will be assessed using Flow-Mediated Dilation (FMD) of the brachial artery, the current gold standard non-invasive technique for evaluating endothelial responsiveness.

A high-resolution ultrasound scanner (add model and manufacturer) with a linear array transducer will be used to image the brachial artery of the dominant arm. Following a 10-minute rest period, baseline artery diameter will be recorded. A pneumatic cuff placed around the forearm will then be inflated to 50 mmHg above participant's systolic blood pressure for five minutes to induce ischaemia.

Upon cuff release, the sudden increase in blood flow (reactive hyperaemia) prompts the endothelium to release nitric oxide, causing vasodilation. The artery will be continuously imaged for three minutes post-deflation, and the peak percentage change from baseline diameter will be calculated as the FMD response. Procedures will follow the internationally accepted guidelines to ensure methodological consistency and minimise measurement bias.

These assessments will be applied in both WP1 and WP2, allowing both cross-sectional and longitudinal analysis of vascular health in pwCF receiving HEMT.

BLOOD TESTS Blood tests will be taken by a health care professional trained in phlebotomy and follow a standard operating procedure for phlebotomy and aseptic technique. A serum-separating tube x 2 (5 ml) and EDTA tube (4ml) will be obtained for each participant (maximum blood volume taken 14ml). Blood test analysis will be performed by Liverpool clinical laboratories and results provided to the clinician- researcher (AC).

The following markers will be analysed:

• Inflammatory markers:

  • C-reactive protein (CRP)
  • Creatine kinase-MB (CKMB) These markers provide insight into systemic inflammation and potential subclinical myocardial stress, both of which are relevant to vascular health.

• Metabolic markers:

  • Glycated haemoglobin (HbA1c) - to assess long-term glycaemic control

  • Full lipid profile, including:

    • Total cholesterol
    • High-density lipoprotein (HDL) cholesterol
    • Low-density lipoprotein (LDL) cholesterol
    • Triglycerides

These biomarkers will be used to investigate the changes over time alongside with vascular function measures (PWV, AIx, FMD). This integrative approach will allow the study to examine how ETI may influence not only vascular function directly but also modifiable cardiometabolic risk factors that contribute to cardiovascular disease development in people with cystic fibrosis.

BLOOD PRESSURE Blood pressure (BP) will be measured using the IEM Mobil-O-Graph. BP readings will be taken in both the supine and upright seated positions to capture potential postural differences and assess autonomic regulation, which can be altered in pwCF. Participants will rest for a minimum of 10 minutes before the first seated BP measurement, followed by supine BP recordings after 2-3 minutes. For each position, three consecutive readings will be taken at one-minute intervals, and the average of the final two will be used in the analysis. This will be completed before PWV measurement.

ECG A resting 12-lead ECG will be performed by a health care professional trained in performing ECGs.

CLINICAL HISTORY A standardised proforma (see Appendix) to collect clinical information relevant to the study will be used. This will be completed at patient investigation visit (for WP1 & 2) The data collected are relevant to the calculation of QRisk3 score and to capture clinical vascular events.

WORK PACKAGE 3 This work package aims to evaluate how well existing CVD risk prediction tools reflect the vascular health status of pwCF receiving CFTR gene modulators. WP3 builds upon the data collected in WP1 and WP2.

After collecting the data from WP1 and WP2, the data will be analysed and used to calculate the QRISK3 score and Framingham Risk Score for each individual's cardiovascular risk. Both scores will be calculated with the existing programme from RStudio.

To assess variability in cardiovascular risk and vascular health across groups and over time, analysis of variance (ANOVA) will be employed. A repeated measures ANOVA will be used to evaluate whether there are significant interactions between time (baseline and 12 months) and group (e.g. pwCF with varying baseline inflammation or metabolic status) on key vascular outcomes, PWV and FMD. This will allow us to explore whether changes in vascular health differ depending on participant characteristics or baseline risk profiles. ANOVA will also be used to compare mean QRISK3 and Framingham scores across different levels of endothelial function or inflammatory status, supporting the exploration of how well these conventional tools align with directly measured cardiovascular health.

This study will be conducted at a tertiary specialist centre for cystic fibrosis care, located in the Northwest of England. All participants with cystic fibrosis (pwCF) will be recruited from outpatient clinics at the Liverpool Heart and Chest Hospital (LHCH), which serves as a regional referral centre for adult CF care. The clinic provides specialist services to a broad geographical catchment area including Merseyside, Cheshire, Lancashire, Greater Manchester, and North Wales, representing a population of approximately 3.5 million people.

The hospital offers routine access to cardiopulmonary testing, phlebotomy services, and a dedicated vascular laboratory, making it a suitable environment for the comprehensive cardiovascular assessments planned within this study.

Healthy control participants will be recruited from the local community, including university staff and students, as well as individuals from surrounding areas. These participants will be assessed either at the clinical laboratories at Liverpool Centre of Cardiovascular Science, located at LHCH.

All study assessments will be conducted in controlled clinical or research environments, with appropriate facilities for phlebotomy, anthropometric measurements, and non-invasive vascular testing. The same equipment and protocols will be used across all visits and locations to ensure consistency and reliability of data collection.

RECRUITMENT:

Participants with CF will be recruited from the CF outpatient clinic at LHCH (face-to-face and telemedicine clinic).

Initial screening will be based on electronic patient records (EPR). Suitable prospective participants may be asked by a treating clinician in NHS clinic appointment whether they wish to participate in the study. If the clinician is not a direct member of the research team, verbal consent will be obtained to pass on patient contact details to a member of the clinical research team (AC). The researcher will contact prospective participants to discuss the study and assess eligibility. Patients will receive an information pack and participation leaflet either in printed format from clinic, in the post, or via email. If an interested prospective participant is seen in an NHS clinic by a clinician-researcher, the information pack and patient information leaflet will be provided to the interested patient during that clinic appointment.

Patients will be given at least 24 hours to consider participation, after which they will be contacted by the researcher (AC) for further discussion, to clarify any questions about the study and to obtain verbal informed consent if they wish to participate. If the patient consents to joining the study, they will be asked to complete the consent form at the investigation appointment for WP1 & 2.

If they wish to withdraw from the study, they may contact any member of the research team. Withdrawing from the study will not impact their normal care and treatment. Contact details of the research team will be provided in the participant information pack.

RECRUITMENT OF HEALTHY PARTICIPANTS The study will be advertised in local GP practices and NHS trust notice boards. Contact information will be provided on the advertisements for prospective healthy controls to approach a member of the clinical-research team (AC). If contacted directly by a prospective participant through advertisement, a member of the clinical-research team will send an information pack and participation leaflet to the prospective participant an information pack and participation leaflet; and will arrange a virtual appointment to check eligibility against the recruitment criteria, discuss the study and answer any questions. A signed consent form will be completed at the investigation appointment.

Prospective participants may have as long as they require to consider participating in the study or not. Participation, or not in the study will not affect their clinical care.

Virtual appointments with the researcher will be made either via telephone or via secure video consultation service (Attend anywhere) as per patient preference.

SCREENING:

Screening for eligibility will be conducted using a combination of clinical record review and on-site assessments. The following procedures will be used to confirm participant eligibility prior to enrolment (Consent will be obtained before screening for healthy controls):

• Routine blood pressure measurement. Individuals with a clinic blood pressure of 140/90 mmHg or higher will be excluded from the study, as this meets the threshold for clinic-measured hypertension according to the National Institute for Health and Care Excellence (NICE) guidelines (NG136)
• Review of recent blood test results (e.g. HbA1c, lipids, CRP), where available from clinical records
• Confirmation of current treatment with CFTR gene modulators (pwCF only)
• Medical history review (e.g. presence of cardiovascular disease)
• 6- led ECG will be performed at screening All data used in initial screening will be obtained from routine care and recent clinical records (electronic patient records, EPR). Laboratory and clinical results will only be used if they were obtained within the last 3 months. If more than 3 months have passed since the most recent tests, new blood samples will be taken as part of the study protocol.

No ionising radiation or biopsies are included in this study. If a participant is found to be ineligible at screening (e.g. due to unexpected clinical findings or recent acute illness), they may be re-screened once, at the discretion of the CI, provided the exclusionary condition is resolved and they continue to meet eligibility criteria. The maximum interval allowed between initial screening and study enrolment will be 8 weeks. Beyond this point, a full re-screening may be required.

CONSENT:

Participants will be provided information about the study by the clinician-researcher in the form of a written information pack and through discussion. All participants will have at least 24 hours to review the material. After this, an appointment will be arranged between the clinician-researcher and participant to discuss the study and answer any questions or queries related to the study. Participants must have capacity to consent to the study and understand written and spoken English. The assessment of capacity to consent to the study will be made by clinician-researcher. Prospective participants may have as much time as they require to consider participating in the study or not within the study time period. If they wish to withdraw their consent or from the study, they may do so at any time without reason or prejudice.

A signed consent form will be completed at the investigation visit for people recruited to WP1&2. At the investigation appointment for WP1&2 the clinician-researcher will discuss the study investigations again and obtain informed consent for the investigations to proceed on the day.

SAMPLE SIZE The sample size for this study is determined using G*Power software (version 3.1). Given the main purpose of the study is descriptive (e.g. describing the CV health of pwCF at baseline) we have powered our study towards the second component of the primary research question, e.g. change over time in pwCF. The stability of FMD% in pwCF is unknown. Previous work by our group (Shelley et al., 2022) reported a mean flow-mediated dilatation (FMD) of 5.3% in people with cystic fibrosis (pwCF), with a 95% confidence interval of -0.98 to 2.88% and a standard deviation of 1.1%.

To detect an absolute difference in 1% change between baseline and 12 months (alpha 0.05, power 0.8) in pwCF with a SD of the differences between paired results of 1.1%, we would require 12 paired samples. To allow for study dropout or incomplete follow up we have pragmatically targeted 16 pairs. 16 pwCF and 16 HC would also give us power of 0.82 to detect an absolute difference of 1% in FMD% between pwCF and HC at baseline.

SAMPLING TECHNIQUE Participants in Group A (pwCF) will be recruited consecutively from the CF outpatient clinics at LHCH. For WP1 and WP2, eligible participants will be approached during routine clinic visits until the required sample size of 16 participants is reached. Although the study uses convenience sampling from a single centre, the CF clinic serves a large and diverse geographical population across Merseyside, Cheshire, Lancashire, Greater Manchester and North Wales. This broad catchment reduces the risk of selection bias.

Healthy control participants (Group B) will be recruited from the community via university postings, local advertisements, and word of mouth. Controls will be age- and sex-matched to CF participants, with a target matching window of ±4 years. Although convenience sampling will be used, efforts will be made to ensure demographic comparability between groups to allow meaningful comparisons of vascular and cardiometabolic health outcomes.

Participants in both groups will be invited to return for follow-up at 12 months (WP2). Only participants who have completed baseline assessments will be invited for follow-up.

Inclusion Criteria:

• Confirmed diagnosis of cystic fibrosis, based on sweat chloride testing and/or CFTR genotyping
• Aged 18 years or older
• Currently receiving CFTR gene modulators, defined as elexacaftor/ tezacaftor/ ivacaftor (ETI), or any next generation gene modulators after ETI, introduced for at least 3 months
• On licensed doses that is listed on Summary of Product Characteristics of each CFTR gene modulator.
• Clinically stable at the time of assessment (no pulmonary exacerbation or hospitalisation in the past 4 weeks)

Inclusion Criteria for healthy control:

• Clinically stable at the time of assessment (no pulmonary exacerbation or hospitalisation in the past 4 weeks)

Exclusion Criteria:

• On long term steroids, or any vasoactive medications
• Diagnosed with end stage organ diseases
• Diagnosed with rheumatoid arthritis, and/or systemic lupus erythematosus
• Pregnancy or breastfeeding
• are a smoker, or have been smoking in the last 10 years
• Inability to undergo vascular assessments (e.g. upper limb vascular anomalies, limb injury)
• Inability to comply with fasting instructions (for blood draws)
• Had an organ transplantation

Participants with CF will be recruited from the CF outpatient clinic at Liverpool Heart and Chest Hospital (face-to-face and telemedicine clinic).

The study will be advertised in local GP practices and NHS trust notice boards, and contact information will be provided on the advertisements for prospective healthy controls to approach a member of the clinical-research team.

Individual participant data sharing is currently undecided. This is due to ongoing consideration of data governance, participant consent provisions, and compliance with UK data protection regulations.